Cell tissue gel containing collagen and hyaluronan

ABSTRACT

Described herein is a cell tissue gel containing collagen and hyaluronan at a weight ratio of 0.01-100:1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patent Ser. No.14/445,944, filed on Jul. 29, 2014, which is a continuation-in-part ofU.S. patent application Ser. No. 12/974,535, filed on Dec. 21, 2010, nowissued as U.S. Pat. No. 8,790,683, which claims priority to U.S.Provisional Patent Application Ser. No. 61/289,132, filed on Dec. 22,2009. The contents of all prior applications are hereby incorporated byreference in their entirety.

BACKGROUND

Stem cell therapy is a promising approach in treating degenerativediseases. However, it remains challenging to retain stem cells at animplantation site and maintain their viability in a recipient so as toaffect tissue repair. There is a need for a vehicle (e.g., a cell tissuegel) that facilitates site-specific stem cell implantation with highcell viability.

Wound healing is a natural restorative response to tissue injury.Healing is the interaction of a complex cascade of cellular events thatgenerates resurfacing, reconstitution, and restoration of the tensilestrength of injured skin. In diabetic patients, wounds can take longerto heal than non-diabetic patients.

SUMMARY

Described herein is a cell tissue gel containing collagen and hyaluronanat a weight ratio of 0.01-100 (collagen): 1 (hyaluronan), e.g.,0.05-100:1, 1-50:1, 100:1, 75:1, 50:1, 30:1, 25:1, 15:1, 10:1, 5:1, 1:1,0.5:1, 0.2:1, or 0.1:1. The concentration of the hyaluronan can be 0.001to 100 mg/ml (e.g., 0.01 to 1 mg/ml, 0.5 to 100 mg/ml, 0.5 mg/ml, 1mg/ml, 1.5 mg/ml, 3 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml 25mg/ml, 30 mg/ml, 50 mg/ml, 75 mg/ml, or 100 mg/ml). The concentration ofcollagen can be 0.001 to 100 mg/ml (e.g., 1-100 mg/ml, 10-100 mg/ml, 100mg/ml, 75 mg/ml, 50 mg/ml, 30 mg/ml, 25 mg/ml, 15 mg/ml, 10 mg/ml, or 5mg/ml). For example, the collagen concentration can be 0.1 to 100 mg/mland the hyaluronan concentration can be 0.01 to 35 mg/ml. In oneembodiment, the collagen concentration is 3 to 40 mg/ml (e.g., 6 mg/mlor 9 mg/ml) and the hyaluronan concentration is 0.2 to 20 mg/ml. Inanother embodiment, the concentration of the hyaluronan is 0.5 to 100mg/ml and the concentration of the collagen is 5 to 50 mg/ml.

The cell tissue gel of this invention can further contain a nutrient forcell growth (e.g., a cell culture medium or a vitamin), a bioactiveagent, one or more matrix factors, and/or stem cells. The bioactiveagent can be a growth factor, e.g., epidermal growth factor, fibroblastgrowth factor, vascular endothelial growth factor, connective tissuegrowth factor, platelet-derived growth factor, insulin-like growthfactor, nerve growth factor, hepatocyte growth factor,colony-stimulating factor, stem cell factor, keratinocyte growth factor,granulocyte colony-stimulating factor, gramulocyte macrophasecolony-stimulating factor, glial derived neurotrophic factor, ciliaryneurotrophic factor, endothelial-monocyte activating polypeptide,epithelial neutrophil activating peptide, erythropoietin, bonemorphogenetic protein, brain-derived neurotrophic factor, BRAK,transforming growth factor beta, and tumor necrosis factor. Exemplarymatrix factors include, but are not limited to, gelatin, fibronectin,elastin, tenacin, laminin, vitronectin, polypeptides, heparan sulfate,chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatansulfate, carrageenan, heparin, chitin, chitosan, alginate, agarose,agar, cellulose, methyl cellulose, carboxyl methyl cellulose, andglycogen. When the cell tissue gel contains gelatin, its concentrationcan range from 0.01-100 mg/ml (e.g., 1 mg/ml). The collagen andhyaluronan concentrations in this gelatin-containing gel can be 6-40mg/ml and 0.2-20 mg/ml, respectively.

Also described is a method of delivering cells (e.g., stem cells) into asubject, including (i) providing a cell implant containing the celltissue gel described above, in which cells grow, and (ii) placing thecell implant in a site of the subject. Also within the scope of thisinvention is use of the cell tissue gel in manufacturing a cell implantused in cell delivery.

The cell tissue gel described herein can be applied to a wound in asubject to treat or promote healing of the wound.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following detailed description of anexample and also from the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a set of two bar graphs showing the effects of various tissuegels containing collagen and hyaluronan on inflammatory cells. CH0—50mg/mL collagen with no hyaluronan; CH5—50 mg/mL collagen+5 mg/mLhyaluronan; CHpma—50 mg/mL collagen+phorbol myristate acetate (PMA). A.activated HL-60 cells. B. U937 cells.

FIG. 2 is a set of two bar graphs (A and B) showing the chemotacticeffects of hyaluronan of various molecular weights. A. HL-60 cells. B.RAW 264.7 cells.

FIG. 3 is a bar graph showing the effects of hyaluronan (HA) of variousmolecular weights at different concentrations on TGF-β1, TGF-β2 andTGF-β3 expressions. The order shown in the legend corresponds to theorder of the bars in each of the three groups, i.e., the first(leftmost) bar is normal medium and the last bar (rightmost) is HA 2590kDa at 5 mg/ml. Comparing to the level for normal medium group, downregulation is denoted as #: p<0.05, ##: p<0.005, ###: p<0.001; upregulation is denoted as *: p<0.05, **: p<0.005, ***: p<0.001.

FIG. 4 is a set of figures (A and B) showing the effects of various celltissue gels on wound healing. Ctrl—open wound; D39—39.5 mg/mL collagen;D40—35 mg/mL hyaluronan; D43—39.5 mg/mL collagen with 3 mg/mLhyaluronan; D44—39.5 mg/mL collagen with 20 mg/mL hyaluronan. 1500 kDahyaluronan was used in this study. A. Images of the wounds. B. Woundsizes.

FIG. 5 is a set of images showing the histology of full-thickness woundmatrices taken at one month (A) and two months (B) post-operation. Thewounds were implanted with various cell tissue gels, control: none, D40:35 mg/mL hyaluronan, D39: 39.5 mg/mL collagen, D43: 39.5 mg/mL collagenwith 3 mg/mL hyaluronan

FIG. 6 is a set of figures (A and B) showing the effects of cell tissuegels on wound healing in diabetic subjects. Control: open wound; Gel-1:50 mg/ml collagen, 5 mg/ml hyaluronan, and 4 mg/ml neomycin; Gel-2: 37.4mg/mL collagen and 35 mg/mL hyaluronan. A. Images of the wounds. B.Wound sizes.

FIG. 7 is a set of pictures showing a porcine wound array model

FIG. 8 is a set of pictures showing wounds on a porcine would arraymodel. All wounds were closed by re-epithelialization. The dark areawithin each lighter rectangle wound area indicates granulation tissuesleft for further healing process. Control—open wound, CH5, 10, 15, 20,30—cell tissue gels containing 50 mg/mL collagen and 5, 10, 15, 20, 30mg/mL hyaluronan, respectively, with 2 mM neomycin.

FIG. 9 is a set of images showing the histology of wounds in the pigmodel. A, two months, B, six months, after the implantation of a celltissue gel.

FIG. 10 is an image showing the histology of healing wounds in the pigmodel.

FIG. 11 is a set of bar graphs showing the effect of collagen (A) andhyaluronan (B) on stem cell proliferation.

DETAILED DESCRIPTION

Described herein is a tissue gel containing collagen and hyaluronan at aweight ratio of 0.01-100 to 1 (e.g., 0.05-100:1, 1-50:1, 100:1, 75:1,50:1, 30:1, 25:1, 15:1, 10:1, 5:1, 1:1, 0.5:1, 0.2:1, or 0.1:1), andoptionally one or more other components such as a matrix factor, abioactive agent, and a nutrient for cell growth.

Collagen

Any of the naturally-occurring collagens or their functional variantscan be used for preparing the tissue gel of this invention. At thepresent time, at least 28 genetically distinct species of collagens havebeen discovered. Collagen can be easily isolated and purified fromcollagen-rich tissues such as skin, tendon, ligament, and bone of humansand animals. Methods for isolating and purifying collagen are well knownin the art. (See, e.g., U.S. Pat. No. 5,512,291; US Patent Publication20040138695; Methods in Enzymology, vol. 82, pp. 33-64, 1982; ThePreparation of Highly Purified Insoluble Collagen, Oneson, I., et al.,Am. Leather Chemists Assoc., Vol. LXV, pp. 440-450, 1970; U.S. Pat. No.6,090,996). Collagen can also be prepared by recombinant technology,such as those described by Advanced Tissue Sciences (La Jolla, Calif.)or purchased from various venders (e.g., Fibrogen; South San Francisco,Calif.). One example follows. Bovine deep flexor tendons, with fat andfascia removed, are washed with water, frozen, and sliced into 0.5 mmslices with a slicer. A suitable amount of the sliced tendons is firstextracted with 50 ml of water at room temperature for 24 hours. Thewater-soluble fraction is discarded and the sliced tendons are thenextracted with an acidic solution (e.g., 0.2 N HCl) at a suitabletemperature (e.g., room temperature) for a suitable period of time(e.g., 12-24 hours). The HCl solution is discarded; the tendons rinsedwith water to remove the residual acid. The rinsed tendons are thenextracted with a basic solution (e.g., 0.75 M NaOH) at a suitabletemperature (e.g., room temperature) for a suitable period of time(e.g., 12-24 hours). After discarding the basic solution, the slicedtendons are neutralized with an acidic solution (e.g., 0.1 N HCl) to apH of 4-7 (e.g. 5) followed by repetitive washes with water to removethe residual base in the tendons. The tendons are then defatted with analcohol (e.g., isopropanol) for a sufficient period (e.g., 16 hours) atroom temperature. The extractant is decanted and the tendons are furtherextracted with an alcohol (e.g., isopropanol) for a suitable period(e.g., 12-24 hours) at room temperature to form a collagen-containingsolution, which can be dried under a clean hood. The collagen powderthus formed can be dispersed in an acidic solution (e.g., 0.5 M or 0.25M acetic acid) in the presence of a proteolytic enzyme (e.g., trypsin orpepsin) and incubated at 4° C. for a suitable period. The mixture isthen filtered through a 100 mesh stainless steel mesh filter and thesolubilized collagen can be precipitated with a 5% NaCl solution. Theprecipitated collagen can be redissolved in the acidic solutiondescribed above and the solution thus formed can be filtered through a100 mesh stainless steel mesh filter to eliminate non-solubilizedparticles. The collagen solution is then dialyzed with distilled waterto remove the acid.

Hyaluronan

The term “hyaluronan” refers to a naturally-occurring anionic,non-sulfated glycosaminoglycan including repeated disaccharide units ofN-acetylglucosamine and D-glucuronic acid, and its derivative.Naturally-occurring hyaluronan (also known as hyaluronic acid orhyaluronate) can be isolated from its natural sources, e.g., capsules ofStreptococci, rooster comb, cartilage, synovial joints fluid, umbilicalcord, skin tissue and vitreous of eyes, via conventional methods. See,e.g., Guillermo Lago et al. Carbohydrate Polymers 62(4): 321-326, 2005;and Ichika Amagai et al. Fisheries Science 75(3): 805-810, 2009.Alternatively, it can be purchased from a commercial vendor, e.g.,Genzyme Corporation, Lifecore Biomedical, LLC and Hyaluron ContractManufacturing. Derivatives of naturally-occurring hyaluronan include,but are not limited to, hyaluronan esters, adipic dihydrazide-modifiedhyaluronan, hyaluronan amide products, crosslinked hyaluronic acid,hemiesters of succinic acid or heavy metal salts thereof hyaluronicacid, partial or total esters of hyaluronic acid, sulphated hyaluronicacid, N-sulphated hyaluronic acid, and amines or diamines modifiedhyaluronic acid. They can be obtained by chemically modifying one ormore of its functional groups (e.g., carboxylic acid group, hydroxylgroup, reducing end group, N-acetyl group). A carboxyl group can bemodified via esterification or reactions mediated by carbodiimid andbishydrazide. Modifications of hydroxyl groups include, but are notlimited to, sulfation, esterification, isourea coupling, cyanogenbromide activation, and periodate oxidation. A reducing end group can bemodified by reductive amination. It also can be linked to aphospholipid, a dye (e.g., a fluorophore or chromophore), or an agentsuitable for preparation of affinity matrices. Derivatives ofnaturally-occurring hyaluronan can also be obtained by crosslinking,using a crosslinking agent (e.g., bisepoxide, divinylsulfone,biscarbodiimide, small homobifunctional linker, formaldehyde, cyclohexylisocyanide, and lysine ethyl ester, metal cation, hydrazide, or amixture thereof) or via internal esterification, photocross-linking, orsurface plasma treatment.

Nutrient for Cell Growth

The term “nutrient” refers to a source of nourishment essential for cellgrowth. It can be an amino acid, vitamin, mineral, carbon source (e.g.,glucose), fatty acid, or a mixture thereof. In one example, the nutrientused in the tissue gel of this invention is a cell growth medium, e.g.,Minimum Essential Medium, Basal Medium Eagle, Dulbecco's ModifiedEagle's medium, Ham's Nutrient Mixtures F-10 or F-12, Medium 199, RPMImedium, Ames' Media, BGJb Medium (Fitton-Jackson Modification), Click'sMedium, CMRL-1066 Medium, Fischer's Medium, Glascow Minimum EssentialMedium, Iscove's Modified Dulbecco's Medium, L-15 Medium, McCoy's 5AModified Medium, NCTC Medium, Swim's S-77 Medium, Waymouth Medium, orWilliam's Medium E.

Bioactive Agent

Any agent (e.g., peptide, polypeptide, oligosaccharide, polysaccharide,or small o molecule) that improves cell viability, promotes cellproliferation, or induces cell differentiation can be used in making thetissue gel of this invention. In one example, the bioactive agent is agrowth factor, such as epidettital growth factor, fibroblast growthfactor, vascular endothelial growth factor, connective tissue growthfactor, platelet-derived growth factor, insulin-like growth factor,nerve growth factor, hepatocyte growth factor, colony-stimulatingfactors, stem cell factor, serotonin, and von Willebrand factor,transforming growth factor, keratinocyte growth factor, granulocytecolony-stimulating factor, granulocyte/macrophage colony stimulatingfactor, glial derived neurotrophic factor, ciliary neurotrophic factor,endothelial-monocyte activating polypeptide, epithelial neutrophilactivating peptide, erythropoietin, bone morphogenetic proteins,brain-derived neurotrophic factor. In another example, the bioactiveagent is a cytokine or chemokine, including, but are not limited to,IL-2, breast-expressed chemokine (e.g., BRAK), kidney-expressedchemokine (e.g., CXCL14). The bioactive agent can also be a celldifferentiation factor, such as dexamethasone, sodium pyruvate, ascorbicacid-2-phosphate, retinoic acid, proline, insulin, transferrin, selenousacid, linoleic acid, and bovine serum albumin, and TGF-β3. In apreferred example, the differentiation factor is a compound thatpromotes chondrogenesis of mesenchymal stem cells (see those disclosedin U.S. Pat. No. 5,908,784), osteogenesis (e.g., dexamethasone, ascorbicacid, (3-glycerol phosphate), adipogenesis (e.g., insulin,isobutyl-methyl xanthine, dexamethasone, indomethacin), cardiomyogenicdifferentiation (e.g., activin A, BMP-4), endothelial celldifferentiation (e.g., EBM-2, dexamethasone, and VEGF), smooth musclecell differentiation (e.g., PDGF-BB), neural induction (e.g., bFGF, EGF,and B27 supplement, DMSO, butylated hydroxyanisole, forskolin, valproicacid, KCl, K252a, and N2 supplement) and endodermal lineagedifferentiation (e.g., dexamethasone, HGF, and FGF-4). The bioactiveagent can also be a Chinese herbal medicine or an active ingredientthereof.

Matrix Factor

A matrix factor is a compound that helps retain cells at an implantationsite. It can be an extracellular factor found in the extracellularmatrix. Examples are, but are not limited to, gelatin, fibronectin,elastin, tenacin, laminin, vitronectin, polypeptides, heparan sulfate,chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatansulfate, carrageenan, heparin, chitin, chitosan, alginate, agarose,agar, cellulose, methyl cellulose, carboxyl methyl cellulose, glycogenand derivatives thereof. In addition, the matrix factor can be fibrin,fibrinogen, thrombin, and polyglutamic acid, a synthetic polymer (e.g.,acrylate, polylactic acid, polyglycolic acid, or poly(lactic-co-glycolicacid), or a cross-linking agent (e.g., genipin, glutaraldehyde,formaldehyde, or epoxide). It is preferred that the matrix factor usedin the tissue gel described herein has a high molecular weight so as toincrease the viscosity of the gel.

Excipient

The cell tissue gel described herein can include one or morepharmaceutically acceptable excipients to provide lubrication andmoisture insulation. The presence of the excipient can also serve as acream to bind and smooth the epithelial layer for healing of shallowwounds.

Lecithin, petroleum jelly, glycerol, glycerine, and glycerin areexemplary excipients that can be included in the cell tissue gel. Otherexcipients are also known and available in the art.

Preparation of Tissue Gels with Cell Embedded

The tissue gel described herein can be prepared by mixing all of itscomponents mentioned above at a desired weight ratio and keeping themixture under suitable conditions to allow gel formation. To prepare acell-embedded gel, desired cells can be mixed with the gel componentsprior to gel formation. The cells can be stem cells obtained from amammal (e.g., bovine, porcine, murine, equine, canine, feline, ovine,simian, and human). Examples are, but are not limited to,placenta-derived stem cells, bone marrow-derived stem cells, stromalcells (e.g., adipose-derived stromal cells), mesenchymal stem cells,tissue progenitor cells, blast cells, or fibroblasts.

The tissue gel thus prepared, with cells embedded, can be implanted to adesired site for tissue repair and other therapeutic purposes.

Wound Healing

The cell tissue gel described herein, with or without cells embedded,can be used to treat or promote healing of a wound (e.g., skin wound).The cell tissue gel can be applied to the wound such that it partially,substantially, or completely covers the wounded area. In particular, thetissue gel can be used to treat a wound in a diabetic patient, The woundcan be shallow wound, a partial wound, or a full-thickness wound.

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference.

Example 1 Stem Cell Viability in Tissue Gels Containing Collagen andHyaluronan

Chorionic villi were obtained from full-term human placentas,mechanically minced, and then digested with colagenase to obtain amaterial containing placenta-derived mesenchymal stem cells. Thematerial was passed through sieves having mesh sizes of 300-, 100-, and37-μm sequentially and then subjected to percoll density gradientcentrifugation to collect stem cells. The cells were suspended in 2×lowglucose Dulbecco's Modified Eagle's Medium (Gibco BRL, LifeTechnologies) supplemented with 20% fetal bovine serum and 200 U/mlgentamycin, at a concentration of 10⁵-10⁷ cells/ml, and mixed withvarious amounts of hyaluronan to form a suspension. Collagen, obtainedfrom porcine dermis, was dissolved in 0.01 N HCl to form a collagensolution having a pH of 4-7. The collagen solution was mixed with thestem cell-containing suspension at an equal volume to form a mixturehaving 6 mg/ml of collagen and 0.05 mg/ml, 0.2 mg/ml, or 0.5 mg/ml ofhyaluronan, or 9 mg/ml of collagen and 0.2 mg/ml of hyaluronan. Mixturescontaining collagen at 6 mg/ml and 9 mg/ml and the same amount of stemcells were used as controls. Any of the above-described mixtures werekept at 37° C. of incubator for 30 minutes to allow collagensolidification so as to form a tissue gel embedded with stem cells. Thetissue gel was cultured at 37° C. with 5% CO₂ supply for up to 14 days.Cell viabilities at different time point (e.g., day 1, day 7, and day14) were examined by trypan blue staining. The results thus obtained areshown in Table 1 below.

As shown in Table 1, the weight ratio of collagen vs. hyaluronan in thetissue gel was critical for maintaining viability of the stem cellsgrown in the gel over time.

TABLE 1 Stem Cell Viability in Tissue Gels Containing Collagen andHyaluronan Concentrations (mg/ml) Percentage of Viable Cells (%)Collagen Hyaluronan Day 1 Day 7 Day 14 6 0 87.5 41.8 40.5 6 0.05 80 72.363.6 6 0.2 85.1 73.2 75.3 6 0.5 70.8 43.4 57.7 9 0 96.3 ± 3.4 N.A. 35.7± 0.3  9 0.2 93.8 ± 1.0 N.A. 72.7 ± 10.4

Example 2 Stem Cell Viability in Tissue Gels Containing Collagen,Hyaluronan, and Gelatin

The stem cell suspension and collagen solution described in Example 1above were mixed at 1:1 by volume together with gelatin (2 or 10 mg/mL)to form stem cell-embedded tissue gels. Their final concentrations ofcollagen, hyaluronan, and gelatin are shown in Table 2 below.

The tissue gels were cultured at 37° C. with 5% CO₂ supply for up to 14days. Cell viabilities at different time point (e.g., day 1, day 7, andday 14) were examined as described in Example 1 above. Table 2 belowlists the percentages of viable cells grown in the tissue gels describedabove.

TABLE 2 Stem Cell Viability in Tissue Gels Containing Collagen,hyaluronan, and Gelatin Concentrations (mg/ml) Percentage of ViableCells (%) Collagen hyaluronan Gelatin Day 1 Day 7 Day 14 6 0 1 93.1 43.554.7 6 0.05 1 87.3 76.8 49.1 6 0.2 1 90.2 71.4 78 6 0.5 1 58.5 55.6 42.46 0 5 80 22.1 41.5 6 0.05 5 93.2 40 46.3 6 0.2 5 71.9 42.9 44.4 6 0.5 544.4 19.1 33.1 9 0.2 1 98.0 ± 0.4 N.A. 47.8 ± 6.5

Example 3 Effects of Tissue Gels Containing Collagen and Hyaluronan onCell Proliferation

It was reported that the characteristics of activated HL-60 by 1.3%dimethyl sulfoxide (DMSO) for 7 days resemble those of activated humanneutrophils that express CD11b and CD32. The activated HL-60 cells wereused to study the effects of cell tissue gels on neutrophilproliferation. Activated HL-60 at 1×10⁴ cells/well in 96-well plate werecultured in RPMI 1640 with cell tissue gels containing variousconcentrations of hyaluronan.

Mouse macrophage cell line RAW 264.7 was confirmed to express macrophageF4/80 and was used to study the effects of cell tissue gels onmacrophage proliferation. RAW 264.7 at 3×10³ cells/well in 96-well platewere cultured in DMEM-10% FBS with the cell tissue gels is containingvarious concentrations of hyaluronan.

NIH-3T3 fibroblasts may represent cells in the stromal layer of tissuesand therefore were chosen to study the effects of cell tissue gels onfibroblast proliferation. NIH-3T3 fibroblasts at 2×10³ cells/well in96-well plate was cultured in DMEM-10% FBS with the cell tissue gelscontaining various concentrations of hyaluronan.

After culturing the above cells in a 37° C. incubator with 5% CO₂ for 3and 5 days, cells were recovered by centrifugation, bursted to exposethe cell nucleus, and stained with Hoechst 33258. The fluorescentintensity was proportional to cell numbers and was measured by aSpectraMax M2e Multi-Mode Microplate Reader with excitation at 365 nmand emission at 460 nm.

The results indicated that the presence of hyaluronan in the cell tissuegels may slightly inhibit neutrophil proliferation during a prolongedperiod, i.e., at or after day 5. No significant effect was observed formacrophage proliferation. The constituents in cell tissue gels atvarious ratios of collagen to hyaluronan promoted fibroblastproliferation both at day 3 and day 5. The results imply a positiveeffects of the cell tissue gels on the process of wound healing.

Example 4 Effects of Tissue Gels Containing Collagen and Hyaluronan onInflammatory Cells

Activated HL-60 cells and U937 cells were used to understand theinteraction of hyaluronan with human neutrophils and macrophages,respectively. Human promyelocytic is HL-60 cells acquired a neutrophilicphenotype after a 7-day DMSO treatment. U937 cells mature anddifferentiate in response to a number of soluble stimuli, adopting themorphology and characteristics of mature macrophages.

In cell culture 24-well plates, cell tissue gels of collagen andhyaluronan at various ratios were individually added to the middle ofthe 1 mm silicone rings. Activated HL-60 cells and U937 cellsrespectively were seeded to the outside of the silicone rings. Aftergelation of the cell tissue gels and the adhesion of the cells, thesilicone rings were removed to allow the migration and infiltration ofcells across the 1 mm distance toward the cell tissue gels. At day 1, 3,and 5, the cell tissue gels were fixed with 4% paraformaldehyde followedwith immunofluorescent staining of nuclear DAPI. The infiltrated cellnumbers were counted using panoramic scanning microscopy.

The cell tissue gels CH0, CH5, and CHpma denote the gels containing 50mg/mL collagen with no hyaluronan, 5 mg/mL hyaluronan, and phorbolmyristate acetate (PMA) respectively. See FIG. 1. The resultsdemonstrated that the presence of hyaluronan in cell tissue gels canstimulate the infiltration of neutrophils and macrophages at early timeperiods. This indicates a positive effect on facilitating the process ofwound healing with the presence of hyaluronan in the cell tissue gels.

Example 5 Chemotactic Effects of Hyaluronan

Boyden chamber with 3 μm pores of membrane was used to study thechemotactic effects of hyaluronan on human neutrophils. 1×10⁵ cells/wellof activated HL-60 cells were placed in the upper chamber and 10 nM fMLP(formyl-methionyl-leucyl-phenylalanine; f-Met-Leu-Phe) or 1 mg/mLhyaluronan at various molecular weights were placed initially at thebottom chamber. Boyden chamber with 5 μm pores of membrane was used tostudy the chemotactic effects of hyaluronan on macrophages. 5×10⁵cells/well of RAW 264.7 cells were placed in the upper chamber and 1μg/mL LPS (lipopolysaccharide) or 1 mg/mL hyaluronan at variousmolecular weights were placed initially at the bottom chamber.

After culturing for 4 or 6 hours, 4% paraformaldehyde was used to fixcells on the membrane and the cells on the upper side were removed bycotton swab. The cells were stained with DAPI and quantitated underlight microscopy.

The results demonstrated a significant chemotactic effect of variousmolecular weights of hyaluronan on activated neutrophils. See FIG. 2. Amolecular weight higher than 780 kDa seems to attract further movementof cells. Since neutrophils play more important roles in clean wounds,these data further indicate a positive effect of hyaluronan in celltissue gels for wound applications.

Example 6 Effects of Hyaluronan on TGF-β1, TGF-β2 and TGF-β3 Expressions

Activated HL-60 cells were cultured in RPMI cultural medium containinghyaluronan at various molecular weights and concentrations. Afterculturing for 3 days, the mRNA expression of TGF-β1, TGF-β2 and TGF-β3were analyzed by quantitative real-time polymerase chain reaction usingappropriate gene detection primers and probes. The relative mRNAexpression levels were normalized with GAPDH (glyceraldehyde 3-phosphatedehydrogenase) expression. The Roche Universal Probe Library System wasused for specific detection of each gene expression and statisticst-test was analyzed for the significacy.

Our results demonstrated that the presence of hyaluronan can inhibitneutrophil expression of TGF-β1 and stimulate the expression of TGF-β3.It was reported that neutralization of TGF-β1 and TGF-β2 or the additionof TGF-β3 to cutaneous wounds not only improve architecture of theneodermis but also reduce scarring. See Shah M, Foreman D, Ferguson M.,J Cell Sci 1995; 108: 985-1002. Our data suggest that cell tissue gelscontaining hyaluronan can result in better healing with reducedscarring.

Example 7 Effects of Cell Tissue Gels on Wound Healing

We established a mouse skin wound model with 8 mm diameter O-ringsutured on the outer of the 6 mm full-thickness circular wound to avoidover contraction of the wound. The advantage of this model is that themode of wound closure is closer to that of human skin in which there-epithelialization and granulation tissue formation are involvedrather than rodent wounds which have excessive contraction during thehealing process.

Four dorsal skin wounds were created on a FVB mouse. Variousformulations of cell tissue gels were implanted in the wounds except anopen control wound. All wounds were covered with medical Tegadenn toprotect from contamination. The closure of wounds were observed and thewound edges were depicted every 2˜3 days. See FIG. 4, A. Image Jsoftware were used to measure the wound sizes at various time pointsalong with the path of wound healing. See FIG. 4, B.

As shown in FIG. 4, B, at day 6, while 78% of wound area remainedunhealed in untreated wound, less than 40% of wound area remainedunhealed in the wounds treated with collagen-hyaluronan cell tissuegels. The results indicated that the cell tissue gels can acceleratewound healing in mice with full-thickness skin wounds.

The neo-tissues at wound area were taken for histology analysis at theend of first and second months post-operation. See FIG. 5. The resultsdemonstrated that the wounds filled with cell tissue gels have restoredthe full-thickness stromal layer before the end of the first month andthe regenerated tissue at the end of the second month was very similarto the peripheral normal skin. The result of the cell tissue gels onstromal layer restoration was very distinct from the wounds just filledwith hyaluronan or collagen alone.

Example 8 Effects of Cell Tissue Gels on Wound Healing in DiabeticSubjects

Diabetic mice were established in strain FVB with intraperitonealinjections of low doses of Streptozotocin (STZ) for 5 consecutive days(50 mg/kg body weight) to induce diabetes in laboratory mice. A weeklater, above 250 mg/dl of blood glucose in mice detected for twoconsecutive days were defined as diabetic mice.

All the animal protocols were pre-approved by the Institutional AnimalCare and Use Committee. The full thickness wounds with 6 mm in diameterwere created in the dorsal skin of mice. An O-ring with 8 mm in diameterwas sutured on the outer of the 6 mm wounds to avoid over contraction ofwounds. Different formulations of cell tissue gels were applied to thewounds and medical Tegaderm were used to protect the wounds fromcontamination. Image J software were used to measure the wound sizes atvarious time points along with the path of wound healing. See FIG. 6.

A month may be required to heal the 6 mm diabetic wounds in comparisonto 13 days for the normal skin wound control. The diabetic wounds werehealed at day 18 to day 25 with the implantation of the cell tissue gelscontaining 20 or 50 mg/mL collagen and 1, 5, 10, 20, 30, or 50 mg/mLhyaluronan. The fastest healing was observed with cell tissue gelsfurther containing vitamins B complex and C. Those wounds healed betweenday 12 to day 20.

Example 9 Effects of Cell Tissue Gels on Wound Healing in a Pig Model

A porcine wound array model was used to quickly screen the effects ofselected cell tissue gels on wound healing. Lanyu pigs at two-month-oldor older were used and the study protocol was approved by the LivestockResearch Institute Animal Use Committee.

At least 20 full-thickness excised wounds were made on either the flankor the back. The efficacy of various formulations of cell tissue gels onwound closure was measured. All wounds were covered with medicalTegaderm to protect from contamination. Wound closures were observed andthe wound edges were depicted every 2-3 days. Image J software were usedto measure the wound sizes at various time points along with the path ofwound healing. See FIG. 7.

At the third day after wound creation, fibroblasts were observed toappear in the subcutaneous fat layer, although the cell number was notlarge and no significant difference among different formulations of celltissue gels was observed. At the fifth day post-operation, fibroblastproliferation and migration toward the granulation tissue becameapparent. Although fibroblasts were still far away from the epithelialside of each wound at the fifth day, the migration distance and cellnumber of fibroblasts were proportional to the amount of hyaluronan inthe cell tissue gel. See Table 3.

TABLE 3 Infiltration distance of Numbers of Group fibroblasts togranulation tissue fibroblasts Open wound + + 50 mg/mL collagen ++ ++ 50mg/mL collagen, +++ +++  5 mg/mL hyaluronan 50 mg/mL collagen, ++++ ++++10 mg/mL hyaluronan 50 mg/mL collagen, ++++ ++++ 15 mg/mL hyaluronan 50mg/mL collagen, +++++ +++++ 20 mg/mL hyaluronan 50 mg/mL collagen, ++++++++++ 30 mg/mL hyaluronan

The appearance of regenerated skin implanted with various cell tissuegels were observed at day 18 post-implantation. See FIG. 8. The celltissue gels at various formulations promoted the healing process andwhich were distinct from the open control.

In contrast to the open control, at month 2, the cell tissue gel groups(as L6 with collagen and hyaluronan) demonstrated a good quality ofhealing in histology that was similar to the normal skin. See FIG. 9, A.At month 6, it was noticed that the thickness and histologicalcharacteristics in epithelial layer were most similar to the normalskin. See FIG. 9, B. The architecture of extracellular matrix, includingcollagen fibrils and elastin, as well as cell numbers and distributionwithin the stromal layer also showed similarity to the normal skin.

The healing efficacy of various tissue gel formulations was alsoanalyzed by histology in the pig model. The formulations that weretested included: L10: 68 mg/mL collagen and 5 mg/mL hyaluronan with 4mg/mL neomycin; L11: 35 mg/mL collagen and 3 mg/mL hyaluronan with 4mg/mL neomycin, and lecithin and glycerin as excipients; L12: 35 mg/mLcollagen and 20 mg/mL hyaluronan with 4 mg/mL neomycin, and lecithin andglycerin as excipients; L13: 35 mg/mL collagen and 10 mg/mL hyaluronanwith 4 mg/mL neomycin, and lecithin and glycerin as excipients; and L14:35 mg/mL collagen and 5 mg/mL hyaluronan with 4 mg/mL neomycin, andlecithin and glycerin as excipients.

Transverse sections were cut through the central part of the wounds,including adjacent uninjured skin and underlying muscular tissue,followed by fixation in buffered formalin and embedding in paraffin.Sections were stained with hematoxylin and eosin and examined in ablinded fashion using light microscopy. The healing quality was furtherexamined through

Masson's and Verhoff s staining for neomatrix regeneration,organization, and elastin fiber formation.

At 6 months post operation, there were no significant differences on thearchitecture of neomatrices between normal skin and the L10˜L14-treatedskin. See FIG. 10. There was a difference between the open controlgroup, which had somewhat thinner collagen fibrils in comparison to thenormal skin.

The excipients made the cell tissue gels worked as a cream to smoothenthe skin if there were shallow cutaneous wounds. There was nosignificant difference in the histology of healed matrices among theL10- and L11˜14-treated groups.

Example 11 Shallow Skin Wounds

For the healing of shallow skin wounds, cell tissue gels were preparedby mixing excipients with collagen (30 mg/mL), 1500 kDa hyaluronan (5,10, 20, 30 mg/mL), with or without vitamins, with or without growthfactors, and antibiotics such as neomycin (4 mg/mL) or anti-microbialpeptide such as pexiganan or its analogs. For example, the contents ofthe above cell tissue gel were mixed with with glycerin and lecithin,and after adjusting the pH to weak-acidic to neutral pH, the mixture wasmixed until a homogeneous gel was formed. The gel can adhered to theskin surface after being applied to the skin.

When such a tissue gel is applied to a full-thickness wound, thepresence of excipients may not promote the healing speed but keep thewounds in a moist and oily state. Such a formulation is more suitablefor partial wounds or first intention wound healing. See Table 4.

TABLE 4 Sample Day 0 Day 7 Day 9 Day 11 Day 13 Day 15 Cell tissue gel100% 61.1% 32.4% 23.4% 13.5% 3.1% with lecithin Cell tissue gel 100%69.0% 34.8% 23.4% 16.2% 2.0% with glycerin Cell tissue gel 100% 68.6%39.9% 21.2% 11.0% 2.2% with glycerin and lecithin

Example 12 Proliferation of Stem Cells

Placenta derived mesenchymal stem cells were culture with cell tissuegels containing collagen and hyaluronan. As shown in FIG. 11, A,collagen had a positive effect on cell proliferation along with time. Asshown in FIG. 11, B, increasing concentrations of hyaluronan in celltissue gels promoted the proliferation of cells; hyaluronan also had apositive effect on cell proliferation along with time. The data suggestthat cell tissue gels support cell growth and can be used in the fieldof regenerative medicine.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A cell tissue gel, comprising collagen andhyaluronan at a weight ratio of 1-50:1, wherein the cell tissue gelpromotes wound healing with reduced scarring and is foamed without across-linking agent.
 2. The cell tissue gel of claim 1, wherein theconcentration of collagen is 1 to 100 mg/ml and the concentration ofhyaluronan is 0.5 to 100 mg/ml.
 3. The cell tissue gel of claim 1,further comprising a nutrient or a bioactive agent.
 4. The cell tissuegel of claim 3, wherein the bioactive agent is a growth factor selectedfrom the group consisting of epidermal growth factor, fibroblast growthfactor, vascular endothelial growth factor, connective tissue growthfactor, platelet-derived growth factor, insulin-like growth factor,nerve growth factor, hepatocyte growth factor, colony-stimulatingfactor, stem cell factor, keratinocyte growth factor, granulocytecolony-stimulating factor, granulocyte macrophage colony-stimulatingfactor, glial derived neurotrophic factor, ciliary neurotrophic factor,endothelial-monocyte activating polypeptide, epithelial neutrophilactivating peptide, erythropoietin, bone morphogenetic protein,brain-derived neurotrophic factor, BRAK, transforming growth factorbeta, and tumor necrosis factor.
 5. The cell tissue gel of claim 3,wherein the nutrient is an amino acid, vitamin, mineral, carbon source,fatty acid, or a mixture thereof.
 6. The cell tissue gel of claim 1,further comprising one or more matrix factors selected from the groupconsisting of gelatin, fibronectin, elastin, tenacin, laminin,vitronectin, heparan sulfate, chondroitin, chondroitin sulfate, keratan,keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin,chitosan, alginate, agarose, agar, cellulose, methyl cellulose, carboxylmethyl cellulose, and glycogen.
 7. The cell tissue gel of claim 6,further comprising a nutrient or a bioactive agent.
 8. The cell tissuegel of claim 1, further comprising a pharmaceutically acceptableexcipient.
 9. The cell tissue gel of claim 8, wherein thepharmaceutically acceptable excipient is lecithin, petroleum jelly,glycerol, glycerine, or glycerin.
 10. The cell tissue gel of claim 1,further comprising an antibiotics or anti-microbial peptide.
 11. Thecell tissue gel of claim 1, wherein the cell tissue gel is produced bymixing all components of the gel to form a mixture, adjusting the pH ofthe mixture to weak-acidic to neutral pH, and mixing the mixture until ahomogeneous gel is formed.
 12. A method of treating a wound in a subjectin need thereof, comprising applying to the wound the cell tissue gel ofclaim
 1. 13. The method of claim 12, wherein the concentration ofcollagen is 1 to 100 mg/ml and the concentration of hyaluronan is 0.5 to100 mg/mL.
 14. The method of claim 12, wherein the cell tissue gelfurther includes a nutrient or a bioactive agent.
 15. The method ofclaim 14, wherein the bioactive agent is a growth factor selected fromthe group consisting of epidermal growth factor, fibroblast growthfactor, vascular endothelial growth factor, connective tissue growthfactor, platelet-derived growth factor, insulin-like growth factor,nerve growth factor, hepatocyte growth factor, colony-stimulatingfactor, stem cell factor, keratinocyte growth factor, granulocytecolony-stimulating factor, granulocyte macrophage colony-stimulatingfactor, glial derived neurotrophic factor, ciliary neurotrophic factor,endothelial-monocyte activating polypeptide, epithelial neutrophilactivating peptide, erythropoietin, bone morphogenetic protein,brain-derived neurotrophic factor, BRAK, transforming growth factorbeta, and tumor necrosis factor.
 16. The method of claim 14, wherein thenutrient is an amino acid, vitamin, mineral, carbon source, fatty acid,or a mixture thereof.
 17. The method of claim 12, wherein the tissue gelfurther includes a pharmaceutically acceptable excipient.
 18. The methodof claim 17, wherein the excipient is lecithin, petroleum jelly,glycerol, glycerine, or glycerin.
 19. The method of claim 12, whereinthe tissue gel further includes an antibiotics or anti-microbialpeptide.
 20. The method of claim 12, wherein the subject is a diabeticpatient.